SummarySheep plasma renin substrate was purified 1,200‐fold by using nephrectomised sheep plasma, followed by DEAE‐Sephadex chromatography and gel filtration. The purified substrate contained 8 μg angiotensin II/mg protein and had an estimated molecular weight of 52,000. The kinetic characteristics of the purified substrate were identical both to those of unpurified nephrectomised sheep plasma and to normal sheep plasma substrates. At pH 7·5, Kmof the human renin‐sheep substrate reaction was 0·29 μM and for sheep renin‐sheep substrate, 2·0 μM. Sheep substrate was susceptible to peptic digestion with generation of pepsitensin.Human renin substrate was less readily purified. DEAE‐Sephadex chromatography of plasma from pregnant women at 36–40 weeks' gestation produced a 70‐fold increase in purity (0·9 μg angiotensin II/mg protein). No further increase was achieved with gel filtration. Human renin substrate behaved as a larger (mol. wt. 82,000) more anionic protein than sheep substrate and was resistant to the proteolytic actions of both pepsin and sheep renin. Kmfor the human renin‐human substrate reaction was high and could not be accurately determined (range 3–8 μM. mean 5·7 μM). The presence of human substrate in a human renin‐sheep substrate system did not alter the measured initial velocity.In both sheep and man, the normal concentration of renin substrate is considerably less than Kmand must therefore be considered a determinant of angiotensin production ratein vivo.